1. Trends in development of stationary phases in chromatography Petr Solich Charles University Czech Republic Separation Techniques, San Francisco, USA - 10 August 2015 CHARLES UNIVERSITY, FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ, CZECH REPUBLIC

2. Faculty of Pharmacy, Charles University, Hradec Kralove Charles University in Prague - founded 1348 - 48 000 students (6.000 foreign) -17 faculties - located in - Prague (14) -Hradec Králové (2) -Pilsen (1) Faculty of Pharmacy in Hradec Králové – founded 1969 - 1500 students (100 foreign) - 11 Departments in 2 buildings - 23 research teams

3. High-performance liquid chromatography  High-pressure pump  Injector – Autosampler  Analytical column (stationary phase)  Detector  Chromatographic software pump autosampler column detector software

4. Recent trends in developments of HPLC  sample preparation (automation, sample volume)  increased separation efficiency (columns)  increased selectivity of metods (detectors)  „Green chromatography“ (low consumption of organic phases)  miniaturization (columns, detectors, instruments) all to increased the laboratory throughput ! FAST CHROMATOGRAPHY

5. Recent trends in developments of HPLC  sample preparation (automation, sample volume)  increased separation efficiency (columns)  increased selectivity of metods (detectors)  „Green chromatography“ (low consumption of organic phases)  miniaturization (columns, detectors, instruments) all to increased the laboratory throughput ! FAST CHROMATOGRAPHY

6. Length - 10 - 250 mm Diameter - 1.0 – 4.6 mm μm Particle size - 1 – 10 μm 100 mm Analytical columns – present situation

7. Length - 10 - 250 mm Diameter - 1.0 – 4.6 mm μm Particle size - 1 – 10 μm 100 mm 3.0 mm Analytical columns – present situation

8. Length - 10 - 250 mm Diameter - 1.0 – 4.6 mm μm Particle size - 1 – 10 μm 100 mm 3.0 mm 3.0 μm Analytical columns – present situation

9.  1. stationary phase based on silica (normal, reversed)  2. stationary phase for HILIC Hydrophilic Interaction Liquid Chromatography) (Hydrophilic Interaction Liquid Chromatography)  3. stationary phases based on zirconium oxide  4. polymeric stationary phase  5. monolithic stationary phase  6. hybrid stationary phases (sub-2-micron phase)  7. core-shell stationary phase Length - 10 - 250 mm Diameter - 1.0 – 4.6 mm μm Particle size - 1 – 10 μm 100 mm 3.0 mm 3.0 μm Analytical columns – present situation

10. Recent trends in chromatographic separation phases - smaller particles (2004) - higher operational pressure - low flow rate - low consumption of MP - short time of analysis - large sequences of samples ? - monolithic rods (2001) - low operational pressure - high flow rate - relatively high consumption of MP - short time of analysis - large sequences of samples High pressure for separation UHPLC Low pressure separation (HPLC, SIA, …) Core-shell technology - porous shell on fused core (2009) - short time of analysis - low consumtion of MP - large sequences of samples ? Sub-2  m columns Monolithic columns Substantial decrease of analysis time !!!

11. S tationary phases - comparison B - Monoliths - Porosity 80 % This special porous character allows relatively high mobile phase flow rates (1–9 ml/min) while keeping the backpressure low.. Monolithic column - made of a highly porous material with two types of pore structure—macropores (size of 2µm) and mesopores (size of 13 nm). J.Sep.Sci., 2009, 32 (15-16) – special issue devoted to Monoliths A - Particle based columns - Porosity 65 %

12. - Comparable speed and effectivity of analysis to sub 2 μm particles in UHPLC, but relatively low back-pressure !! - Shorter diffusion path compared to conventional total porous particles - RP-Amide, Phenyl-Hexyl, HILIC, F5, ES-Cyano - pressure cca 600 psi, pH range 2 – 9 Ascentis, Kinetex, Poroshell, Halo… Core-shell (Porous shell) particle columns Solid core

13. sub-2-microne porous shell – 20% w 1/2 3- 5 µm CORE-SHELL PARTICLES – MASS TRANSFER

14. Bioanalytical applications of monolithic columns

15. Bioanalytical laboratory Charles University, Teaching Hospital, Hradec Kralove

16. Biologically active compounds analysed in Bioanalytical laboratory in hospital using monolithic columns  Retinoic acid in serum  Neopterin in urine  Retinyl esters = Vitamin A estersin serum  Vitamin A, E, D in human serum in erythrocytes in lipoprotein fractions Application: - in clinical monitoring of patients with cardiovascular problems, - in monitoring of oncological treatment (side effects, nutritional status - in treatment of metabolic diseases (diabetes mellitus, pancreatitis) - clinical monitoring of elderly patients (level of antioxidants) - monitoring of patients with nephorological infections

17. Bioanalytical applications of monolithic columns Determination of retinoic acids (RA)

18. Most important RA are 13-cis RA, all-trans RA For treatment of hemato-oncological deseases, a formulation Tretinoin is used (contain RA) tzv. Retinoidal syndrom – found in many patients (about 25 %) treated by Tretinoin Retinoidal syndrom: - high temperatures of body - acute pulmonary complications - hypotensis - eodems need for monitoring of RA in patients treated with formulation Tretinoin

19. HPLC method for determination of RA, vitamin A (retinol) and vitamin E (tocopherols) in serum SPE Extraction: IS (20 µmol.L -1 ) 250 µl of serum Deproteination by ethanol (650 μl, 8 min, 4 ºC). centrifugation (2 000 ×g, 15 min, 4 ºC) supernatant placed on SPE colunmn Elution: by hexan (2 000 μl) Evaporation to dryiness - 35 ºC Addition of 250 μl of methanolu HPLC determination SP: monolithic column Chromolith Performance RP-18e, 100×4.6mm MP: ACN : 1% ammonium acetate 95:5 (v/v) 4 min flow rate 1.5 ml.min -1, 3 min flow rate 3.2 ml.min -1 („flow rate“ gradient !!!) injection 20 μl DAD detection: of retinol, all-trans- and 13-cis RA 325 nm, alfa-, gama- tokoferol a tocol 295 nm Time of analysis - 7 min

20. Determination of retinoic acids and vitamins A and E in human serum Krčmová L, Urbánek L, Solichová D, Kašparová M., Vlčková L., Melichar B, Sobotka L., Solich P: J. Sep. Sci. 32 (15-16), 2009, 2804-2811 4 min - flow rate 1.5 ml.min-1, 3 - min flow rate 3.2 ml.min-1

21. Determination of Vitamin D (+ vitamins A and E) Bioanalytical applications of monolithic columns

22. Determination of Vitamin D Common name for a group of 9,10-secosteroids Most important – D 3 – cholecalciferol – animal origin created in organism by UV radiation from provitamin D3 D 2 –ergocalciferol – plant origin Both forms have the same activity in organism Vitamin D is transformed to active form by hydroxylation Influence on metabolism of phosphorus and calcium in the body Influence on calcium level in bones Prevention of osteoporosis

23. Development of HPLC method for simultaneous determination of vitamines A, E and D Vitamin D : Ergocalciferol (D 2 ) Cholecalciferol (D 3 ) metabolite calcidiol - 25(OH)D 3 metabolite calcitriol - 1,25(OH)D 3 3 Vitamin E – α-tocopherol Concentration in serum 19 – 35 μmol/L Vitamin A - retinol Concentration in serum 1,05 – 2,27 μmol/L

24. Chromolith RP-18e, 100×4.6mm + 50×4.6 mm Development of HPLC method for A, E and D standards

25. Spiked serum Sklenářová H., Koblová P., Chocholouš P., Šatinský D., Krčmová L., Kašparová L., Solichová D., Solich P., Analytical Letters 44(1-3), 2011, p. 446-456. Development of HPLC method for A, E and D

26. Determination of neopterin in urine Bioanalytical applications of monolithic columns

27. reflect the stage of activation of the cellular immune system and pathogenesis and progression of various diseases: - in viral infections (HIV…) - in autoimmune or inflammatory diseases - rejection episodes following allograft transplantation - in several malignant diseases - neopterin concentrations are very closely linked with the progression of these diseases ! Therefore it is of interest for laboratory diagnosis to measure the degree of activation of the human immune system. This is possible in an easy but specific way by the determination of neopterin concentrations. Neopterin = a diagnostic marker

28. Chromatogram of urine Chromolith RP-18 e, 100 x 4.6 mm, flow rate 1,2 ml/min Uric acidkreatinin neopterin kynurenin

29. Monolithic stationary phases - trends HR-monolithic columns ( introduced 2011) Higher separation performance - increased by 50% (System pressure increased by 60% ) Performance similar as 2.7 µm core-shell particle columns Currently – increasing choice (C18, C8, SILICA, NH2, CN, PHENYL) Size - length 5 – 100 mm, diameter 2.0 - 4.6 mm Working range of pH 2 – 7.5 (only)

30. Applications of sub-2-micro columns Determination of pharmaceuticals by UHPLC

31. Particles 5 μm Human hair 60 μm Particles 1.7 μm Columns with 1,7 μm particles hybrid sorbent X-Bridge (BEH-Bridged Ethylene Hybrid ) - particles 1.7 µm - range pH - 1- 12 - pressure up to 1000 bar

32.  working up to 100 MPa (15 000 Psi, 1000 bar)  pressure resistant connections  X-Bridge sorbents  particle size 1.7 μm  special injection system  acquisition rate 80 points/s  special optical cell (500 nl, 10 mm) Ultra High Performance Liquid Chromatography (UHPLC)

33. Time of analysis: 16.3 min Solvent consumption: 11.4 ml Time of analysis: 2.3 min Solvent consumption 1.1 ml Classical C18 column 5 um, 125 x 4.6 mm X-Bridge column 1.7 um, 50 x 2.1 mm Nováková L, Solich P.: J.Sep.Sci. 29, 2006, 2433 Speed acceleration 7 x HPLC UPLC 0 4 8 12 16 min 0 0,5 1 1,5 2 min Application of UHPLC – Diclophenac gel

34. Bioanalytical applications of sub-2-micro columns Determination of phenolic compounds by UHPLC

35. UHPLC separation of 30 phenolic compounds Acquity BEH C18 (100 x 2.1 mm/ 1.7 μm), gradient 0.1 % formic acid/MeOH/ from 88:12 to 30:70 (v/v), f.rate 0.45 ml/min, UV - 280 nm, 25° C Phenolic acids Catechines Coumarins Flavonoids Nováková L., Spáčil Z., Seifrtová M., Solich P.: Talanta 80, 2010, 1970-1979

36. Bioanalytical applications of core-shell columns Determination of vitamin A and E

37. suitable for UHPLC system suitable for HPLC and UHPLC system suitable for HPLC system ! ! Kučerová B., Krčmová L., Solichová D., Plíšek J., Solich P.: Comparison of new high-resolution monolithic column with core-shell and fully porous columns for THE analysis of retinol and α- tocopherol in human serum and breast milk BY UHPLC, J. Sep. Sci. 23, 2013, p.2223-2230 Comparison of core-shell and HR monolithic columns – for determination of vitamns A and E

38. Comparison of chromatographic separation Classical particulate C18 150 x 4.6 mm; 5 μm Monolithic column 100 x 4.6 mm UHPLC – BEH silica 100 x 2.1 mm; 1.7 μm Determination of retinol and alpha-tocopherol in human serum

39. Where is the future ??? X sub-2  m column monolithic columns X Very high pressure separation Two opposite trends Low pressure separation Substantial decrease of analysis time !!! !? Combination of high pressure using monolithic design !? !? Combination of all features descibed !? core-shell design X high-temperature LC

40. Conclusions Applications of modern technologies (combination of modern HPLC/UHPLC method, new stationary phases with quick and easy sample preparation method) for (bioanalytical) applications allow:  Faster and robust analyses  Large sequences  Small volumes of samples  Small volumes of solvents  No environmental pollution  Cheap  Increase laboratory throughput

41. Acknowledgement

42. Thank you for your attention